Direct and continuous root alone or root/shoot production from transgenic events derived from green regenerative tissues and its applications

ABSTRACT

The present invention provides assays and methods for efficiently testing a polynucleotide of interest for a phenotype in a root. In some embodiments, the assays and methods include regenerating green tissue that is transgenic for at least one polynucleotide of interest into one or more transgenic plantlets that have at least one transgenic root. Further provided are methods of making a root assay by contacting green tissue with a first rooting medium to produce a plantlet and a plurality of roots. Additionally provided are methods of assaying for insecticidal activity on a live root. Accordingly provided herein is a substantially contamination-free, root bioassay. Further provided are methods of identifying a promoter having activity in a root.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/291,704, filed Dec. 31, 2009, which is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of genetic manipulation of plants; inparticular, the invention provides assays and methods for efficientlytesting a polynucleotide of interest for a phenotype in a plant tissuesuch as a plantlet, a root or a leaf.

BACKGROUND OF THE INVENTION

The economic value of roots arise not only from harvested roots, butalso from the ability of roots to alter the soil in which they grow andto funnel nutrients to support growth and increase vegetative material,seeds, fruits, etc.

Roots have four main functions. First, they anchor the plant in thesoil. Second, they facilitate and regulate the molecular signals andmolecular traffic between the plant, soil and soil fauna. Third, theroot provides a plant with nutrients gained from the soil or growthmedium. Fourth, they condition local soil chemical and physicalproperties. Roots arise from meristems cells that are protected by aroot cap during root elongation, but as the root grows out, the capcells abscise and the remaining cells differentiate to the tip.Depending on the plant species, some surface cells of roots can developinto root hairs. Some roots persist for the life of the plant, othersgradually shorten as the ends slowly die back and some may cease tofunction altogether due to external influences.

Because plants are sessile organisms, their survival is criticallydependent on rapid adaptation to environmental changes. In the soil,change can arise from alteration of the concentration of oxygen orcarbon dioxide, nutrient availability, the presence (or absence) ofmicroorganisms and overall soil humidity. For example, oxygen levels inthe rhizosphere decrease rapidly during flooding. Hypoxic or anoxicconditions occur in submerged plant tissues and can have lasting effectson the subsequent growth and/or development of the plant.

Roots are also the sites of intense chemical and biological activitiesand as a result can strongly modify the soil they contact. For example,roots secrete a wide variety of high and low molecular weight moleculesinto the rhizosphere in response to biotic and abiotic stresses. Theyare also capable of absorbing toxic substances from the soil and thenstoring or modifying the toxins, resulting in soil improvement.

Roots coat themselves with surfactants and mucilage to facilitate thesetypes of activities. Specifically, roots attract and interact withbeneficial microfauna and flora that help to mitigate the effects oftoxic chemicals, pathogens and stress in addition to facilitating waterand nutrient assimilation and mobilization. Nutrients can take the formof ions and organic and inorganic compounds. Uptake of nutrients byroots produces a “source-sink” effect in a plant. The greater the sourceof nutrients, the larger “sinks” (such as stems, leaves, flowers, seeds,fruits, etc.) can grow.

Currently, transient gene expression has been applied to dicot speciesusing the hairy root system to do a quick gene testing in roots, butestablishing the hairy root system for maize and delivering transgenesin roots using Agrobacterium rhizogenes has been difficult. Generatingtransgenic maize plants with a callus tissue system by standardprotocols uses a whole cycle of the transformation process which is atime-consuming process.

To date, there is only limited ability to efficiently and quickly testgenes and root promoters in vivo in a root, for example, to assess thestrength of a promoter in a root, to assess a gene's effect on thetolerance of roots to pests that attack roots (e.g., insects, fungi,bacteria, viruses, or nematodes) or to assess a gene's effect on thenutritional composition of roots for human food or animal feedapplications. Thus a need exists for a highly efficient way to testpolynucleotides in the root of a plant and generate plants expressingthem in the root.

SUMMARY OF THE INVENTION

Compositions and methods are provided for efficiently testing apolynucleotide of interest for a phenotype in a plant tissue. While theinvention is primarily discussed with respect to the root, it isrecognized that the leaf, the plantlet, or other tissues may be used inthe methods of the invention. More specifically, the embodiments of thepresent invention relate to assays and methods of regenerating greentissue into one or more plantlets that have at least one root. In someexamples, the green tissue is transgenic for at least one polynucleotideof interest and the green tissue is regenerated into one or moretransgenic plantlets having at least one transgenic root. The root,plantlet, or leaf, non-transgenic or transgenic, may be optionallysubjected to a biotic stress, pest, or pathogen. The plant tissue may beassayed for one or more phenotypes. Such root phenotypes include but arenot limited to increased root size, increased overall root mass, alteredroot architecture, increased expression level of mRNA or protein,increased biochemical content, increased tolerance or resistance to apest or pathogen, modulation in biotic mass of the root, modulatedyield, such as increased yield, as compared to the correspondingphenotype of a control. Similar phenotypes can be assessed for the leafand plantlet.

Also provided herein are methods of making a root assay by contactinggreen tissue with a first rooting medium to produce a plantlet. Therooting medium may be a liquid, gel, or solid medium, including, forexample, a medium gelled with agar or an agar substitute. The plantlethas at least one root that is removed from the medium and is contactedwith a second rooting medium to produce a plurality of roots. In someembodiments, the rooting medium lacks agar or an agar substitute

Additionally, a method of assaying for insecticidal activity on a liveroot is provided herein. The method includes regenerating green tissueinto one or more plantlets comprising at least one live root. In someembodiments, the green tissue is transgenic for a polynucleotide ofinterest. The at least one root of the plantlet is contacted with arooting medium. The root is exposed to one or more pests to infest themedium for infestation. In some embodiments, the medium and pest aresubstantially free of contamination. A phenotype of the root and/or pestis determined.

Accordingly, one of the embodiments includes a substantiallycontamination-free, root bioassay. The bioassay includes a live monocotplantlet with at least one live root. In some examples, the plantlet hasat least one live root that is transgenic for a polynucleotide ofinterest. The root is placed in culture dish. The dish includes arooting medium that contacts the root of the plantlet.

Methods of identifying a promoter having activity in plant tissue,particularly the root, are also provided. The methods relate toregenerating green tissue transgenic for a promoter of interest operablylinked to a polynucleotide into one or more stably transformedtransgenic plantlets. The plantlets have at least one live transgenicroot. Further encompassed by the methods is determining whether thepolynucleotide is expressed in root cells of the plantlet. The relativestrength of a promoter in a root cell, the spatial expression of apromoter in the root, or whether the promoter is a root-preferredpromoter may also be evaluated if desired. The methods may includedetermining the expression level of the polynucleotide and/orpolypeptide encoded by the polynucleotide in root cells of the plantlet.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flow chart demonstrating an efficient screening schemeusing in vitro bioassay plantlets.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting. The following ispresented by way of illustration and is not intended to limit the scopeof the invention.

Indeed, the invention may be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will satisfyapplicable legal requirements.

Many modifications and other embodiments of the invention set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions. Therefore, it is to be understood that theinvention are not to be limited to the specific embodiments disclosedand that modifications and other embodiments are intended to be includedwithin the scope of the appended claims. Although specific terms areemployed herein, they are used in a generic and descriptive sense onlyand not for purposes of limitation. The articles “a” and “an” are usedherein to refer to one or more than one (i.e., to at least one) of thegrammatical object of the article. By way of example, “an element” meansone or more than one element.

As used herein, the term “transgenic” means a plant or plant cell orplant part (e.g., a plant tissue or a plant organ) that comprisesgenetic material additional to the naturally occurring nucleic acidwithin the plant, cell or part. For example, the genome of a transgenicplant or plant cell or plant part may comprise nucleic acid from adifferent organism such as an animal, insect, bacterium, fungus ordifferent plant species or variety. Alternatively, the genome of atransgenic plant or plant cell or plant part may comprise one or moreadditional copies of nucleic acid that occur naturally in the same plantspecies or variety. Alternatively, the genome of a transgenic plant orplant cell or plant part may comprise nucleic acid that does not occurin nature e.g., RNAi. The genome of a transgenic plant or plant cell orplant part may also contain a deletion relative to the genome of anisogenic or near-isogenic naturally-occurring plant e.g., as a result ofhomologous recombination or recombinase-induced recombination.

As used herein, the term “green tissue” refers to green regenerativetissue or green callus tissue which is green, shiny, nodular and compactas compared to monocot plant callus tissue. Green tissues areorganogenic and have meristem-like structures. See U.S. Pat. No.7,102,056, incorporated by reference in its entirety.

The term “root-preferred” is intended to mean that expression of theheterologous polynucleotide sequence is most abundant in the root. Whilesome level of expression of the heterologous nucleotide sequence mayoccur in other plant tissue types, expression occurs most abundantly ina cell of the root or in a type of root, which may include, but is notlimited to primary, lateral, and adventitious roots.

The term “root” is intended to mean any part of the root structure,including but not limited to, the root cap, apical meristem, protoderm,ground meristem, procambium, endodermis, cortex, vascular cortex,epidermis, and the like.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers.

Accordingly, an “enhancer” is a nucleotide sequence which can stimulatepromoter activity and may be an innate element of the promoter or aheterologous element inserted to enhance the level or tissue-specificityof a promoter. Promoters may be derived in their entirety from a nativegene, or be composed of different elements derived from differentpromoters found in nature, or even comprise synthetic nucleotidesegments. It is understood by those skilled in the art that differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions. Promoters which cause a nucleic acidfragment to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”.

The term “dim light” refers to light that is approximately 5 to 50 μEm⁻²s⁻¹.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

Previously a whole cycle of the transformation process was used togenerate transgenic maize plants with a callus tissue system, but thisis a time-consuming process. Transient gene expression has been appliedto dicot species using the hairy root system to do a quick gene testingin roots, but establishing the hairy root system for maize andexpressing transgenes using Agrobacterium rhizogenes have beendifficult. The current invention utilizes a highly regenerative tissuesystem which can produce transgenic organogenic tissues ready for shootregeneration and root formation. For example, using a visible marker,transgenic sectors can be easily identified under a fluorescencemicroscope and used for fast and continuous root production as well asshoot regeneration from transgenic green tissues by placing themdirectly on the rooting medium. Green tissues are more organogenic thancallus tissues, and advantageously these green tissues can be maintainedfor long periods with a minimal loss of regenerability. This is incontrast to the rapid loss of regenerability that occurs when using astandard callus tissue system. A further advantage from practicing themethods and bioassays described herein is that, because green tissue isused, multiple plants can be produced from the same transgenic eventduring an extended time period.

Accordingly, provided herein are methods and assays for efficientlyidentifying the affects of expression of one or more polynucleotides ofinterest on plant tissues, particularly on the roots, transgenic for theone or more polynucleotides. The plant tissues are then analyzed forexpression of the polynucleotides. Such affects or phenotypes for rootsinclude, but are not limited to, modulated root size, overall root mass,root architecture, expression level of mRNA or protein, biochemicalcontent of the root, tolerance to a biotic stress, tolerance orresistance to a pest, tolerance or resistance to a pathogen, yield,agronomic traits, increased disease resistance, nutritional enhancement,and the like. Also provided herein are methods and assays forefficiently determining whether a live plantlet or root has endogenousresistance or susceptibility to a biotic stress, such as a pest orpathogen. This would be of interest when screening germplasm usingnon-transgenic germinating plantlets/roots. In one example,polynucleotides effective for preventing corn root worm infestation ordamage to roots associated with corn root worms may be identified usingthe provided methods and assays. In another aspect, the provided methodsand assays may be used to determine whether a promoter is functional ina root cell, the relative strength of a promoter in a root cell, thespatial expression of a promoter in the root, or whether the promoter isa root-preferred promoter. In addition, the methods and assays describedherein can be applied for rapid production of non-transgenic monocotplants or transgenic monocot plants. Efficient regeneration of plantswould facilitate the study of plants with improved traits or phenotypes.

In one aspect, the methods include regenerating green tissue into one ormore plantlets having at least one root. In some examples, the greentissue is transgenic for at least one polynucleotide of interest andgives rise to a transgenic plantlet having at least one transgenic root.In other applications, the green tissue can be used to produce rootcultures, for example, transgenic root cultures. The polynucleotide ofinterest may be any suitable polynucleotide and may be either endogenousor heterologous to the plant cell being transformed. Polynucleotidesencompass all forms of nucleic acid sequences including, but not limitedto, single-stranded, double-stranded, triplexes, linear, circular,branched, hairpins, stem-loop structures, branched structures, and thelike. In some instances, the polynucleotide of interest may encode apolypeptide of interest which is expressed in the cell. Thepolynucleotide of interest may confer a particular trait of interest tothe plant, for example, such as, but not limited to disease resistanttraits, insect resistant traits, nutritional enhancements, agronomictraits, firmness, acidity content, sugar content, texture, oil, starch,carbohydrate, or nutrient metabolism, increased oil production,increased protein production, unique oil and protein production,increased fermentable starch production, increased content of essentialamino acids, increased content of fatty acids and the like. Thepolynucleotide of interest may be thioredoxin (Cho et al. 1999, ProcNatl Acad Sci USA 96: 14641-14646), lactoferrin, or lysozyme (Humphreyet al. 2002, J of Nutrition 32(6): 1214-1218). In one example, thepolynucleotide of interest is a selectable or screenable marker gene.Exemplary marker genes are described elsewhere herein. In someinstances, the polynucleotide of interest may suppress the expression ofa target molecule in the plant cell, for example, Ca²⁺-dependent proteinkinase1 (CDPK1), Plant Cell 17:2911-2921 (2005); Arabidopsis Ran bindingprotein, AtRanBP1c, Plant Cell, 13: 2619-2630 (2001). The inhibitorypolynucleotide may any suitable polynucleotide including but not limitedto miRNA, a siRNA, dsRNA, an antisense polynucleotide and the like.

In one embodiment, recombinant vectors including one or morepolynucleotides of interest suitable for the transformation of plantcells are prepared. These may be used to construct a recombinantexpression cassette which can be introduced into the desired plant cell.In one example, an expression cassette will typically comprise apolynucleotide of interest operably linked to a promoter sequence andother transcriptional and translational initiation regulatory sequenceswhich are sufficient to direct the transcription of the polynucleotidesequence in the intended tissues (e.g., entire plant, leaves, roots,etc.).

A number of promoters can be used in the practice of the presentinvention. The promoters can be selected based on the desired outcome.That is, the nucleic acids can be combined with constitutive, inducible,tissue-preferred, root-preferred promoters or other promoters forexpression in the explant, green tissue, root, or regenerated plant.

Constitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in WO 99/43838and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al.(1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters include, for example, thosedisclosed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maize1n2-2 promoter, which is activated by benzene sulfonamide herbicidesafeners; the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides; andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters. See, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257 and thetetracycline-inducible and tetracycline-repressible promoters forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156, herein incorporated by reference.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller et al. (1991) Plant Cell 3(10):1051-1061 (root-specific controlelement in the GRP 1.8 gene of French bean); Sanger et al. (1990) PlantMol. Biol. 14(3):433-443 (root-specific promoter of the mannopinesynthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al.(1991) Plant Cell 3(1): 11-22 (full-length cDNA clone encoding cytosolicglutamine synthetase (GS), which is expressed in roots and root nodulesof soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633-641,which discloses two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa. The promoters ofthese genes were linked to a beta-glucuronidase reporter gene andintroduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach et al. (1991) describe their analysis ofthe promoters of the highly expressed rolC and rolD root-inducing genesof Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76).They concluded that enhancer and tissue-preferred DNA determinants aredissociated in those promoters. Teeri et al. (1989) EMBO J. 8(2):343-350used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, which is an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene. The TR1′ gene, fused to nptII (neomycinphosphotransferase II), showed similar characteristics. Additionalroot-preferred promoters include the VfENOD-GRP3 gene promoter (Kusteret al. (1995) Plant Mol. Biol. 29(4):759-772); the ZRP2 promoter (U.S.Pat. No. 5,633,636); the IFS1 promoter (U.S. patent application Ser. No.10/104,706) and the rolB promoter (Capana et al. (1994) Plant Mol. Biol.25(4):681-691). See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,459,252;5,401,836; 5,110,732; and 5,023,179.

A strongly or weakly constitutive plant promoter that directs expressionof a polynucleotide of interest nucleic acid in all tissues of a plantcan be employed. Such promoters are active under most environmentalconditions and states of development or cell differentiation. Inaddition to the promoters mentioned above examples of constitutivepromoters include the 1′- or 2′-promoter of Agrobacterium tumefaciens,and other transcription initiation regions from various plant genesknown to those of skill. Where over expression of a polypeptide ofinterest is detrimental to the plant, one of skill will recognize thatweak constitutive promoters can be used for low-levels of expression.Generally, by “weak promoter” a promoter that drives expression of acoding sequence at a low level is intended. By “low level” levels fromabout 1/1000 transcripts to about 1/100,000 transcripts, to about as lowas 1/500,000 transcripts per cell are intended. Alternatively, it isrecognized that weak promoters also include promoters that are expressedin only a few cells and not in others to give a total low level ofexpression. Where a promoter is expressed at unacceptably high levels,portions of the promoter sequence can be deleted or modified to decreaseexpression levels. In those cases where high levels of expression is notharmful to the plant, a strong promoter, e.g., a t-RNA, or other pol IIIpromoter, or a strong pol II promoter, e.g., the cauliflower mosaicvirus promoter, CaMV, 35S promoter can be used.

Alternatively, a plant promoter can be under environmental control. Suchpromoters are referred to as “inducible” promoters. Examples ofenvironmental conditions that may alter transcription by induciblepromoters include pathogen attack, anaerobic conditions, or the presenceof light. In some cases, it is desirable to use promoters that are“tissue-specific” and/or are under developmental control such that thepolynucleotide of interest is expressed only in certain tissues orstages of development, e.g., leaves, roots, shoots, etc. Promoters ofgenes related to pesticide resistance and related phenotypes may also beused.

Tissue specific promoters can also be used to direct expression ofheterologous structural genes, including polynucleotides of interest.Thus, the promoters can be used in recombinant expression cassettes todrive expression of any gene whose expression is desirable in thetransgenic plantlets. Similarly, enhancer elements, e.g., derived fromthe 5′ regulatory sequences or intron of a heterologous gene, can alsobe used to improve expression of a heterologous structural gene.

In general, the particular promoter used in the expression cassette inplants depends on the intended application. Any of a number of promoterswhich direct transcription in plant cells can be suitable. In additionto the promoters noted above, promoters of bacterial origin whichoperate in plants include the octopine synthase promoter, the nopalinesynthase promoter and other promoters derived from T1 plasmids. See,Herrera-Estrella et al. (1983) Nature 303:209. Viral promoters includethe .sup.35S and 19S RNA promoters of CaMV. See, Odell et al. (1985)Nature 313:810. Other plant promoters include theribulose-1,3-bisphospha- the carboxylase small subunit promoter and thephaseolin promoter. The promoter sequence from the E8 gene (see, Deikmanand Fischer (1988) EMBO J. 7:3315) and other genes are also favorablyused. Promoters specific for monocotyledonous species are alsoconsidered (McElroy and Brettell (1994) “Foreign gene expression intransgenic cereals” Trends Biotech. 12:62-68.) Alternatively, novelpromoters with useful characteristics can be identified from any viral,bacterial, or plant source by methods, including sequence analysis,enhancer or promoter trapping, and the like, known in the art.

In preparing expression vectors, sequences other than the nativepromoter of the polynucleotide of interest may also be used. If properpolypeptide expression is desired, a polyadenylation region can bederived from the native gene, from a variety of other plant genes, orfrom T-DNA. Signal/localization peptides, which, e.g., facilitatetranslocation of the expressed polypeptide to internal organelles (e.g.,chloroplasts) or extracellular secretion, can also be employed.

The vector can include a selectable or screenable marker gene as, or inaddition to, a particular polynucleotide of interest to provide orenhance the ability to identify transformants by conferring a selectablephenotype on the transformed plant cells. “Marker genes” are genes thatimpart a distinct phenotype to cells expressing the marker gene and thusallow such transformed cells to be distinguished from cells that do nothave the marker. Such genes may encode either a selectable or screenablemarker, depending on whether the marker confers a trait which one can“select” for by chemical means, i.e., through the use of a selectiveagent (e.g., a herbicide, antibiotic, or the like), or whether it issimply a trait that one can identify through observation or testing,i.e., by “screening”, e.g., bar, pat, GAT, PMI, hpt, nptII, DS-RED, GFP,YFP, GUS. Of course, many examples of suitable marker genes are known tothe art and can be employed in the methods and assays. Marker genes mayalso be used to monitor gene expression and protein localization inplant cells, such as root cells via visualizable reaction products or bydirect visualization of the gene product itself. Accordingly, manyselectable marker coding regions may be used in connection with apromoter. Examples of selectable markers include nptII. (Potrykus etal., 1985), which provides kanamycin resistance and can be selected forusing kanamycin, G418, paromomycin, etc.; bar, which confers bialaphosor phosphinothricin resistance; a nitrilase such as bxn from Klebsiellaozaenae which confers resistance to bromoxynil (Stalker et al., 1988)and a mutant acetolactate synthase (ALS) which confers resistance toimidazolinone, sulfonylurea or other ALS inhibiting chemicals (EuropeanPatent Application 154,204, 1985) and a methotrexate resistant DHFR(Thillet et al., 1988). Such vectors also generally include one or moredominant selectable marker genes, including genes encoding antibioticresistance (e.g., resistance to hygromycin, kanamycin, bleomycin, G418,streptomycin, paromomycin, or spectinomycin) and herbicide-resistancegenes (e.g., resistance to phosphinothricin acetyltransferase orglyphosate) to facilitate manipulation in bacterial systems and toselect for transformed plant cells.

A number of techniques and protocols may be used to produce greentissue. For example, when it is desired that green tissue be transgenicfor a polynucleotide of interest, the green tissue itself may betransformed using conventional methods, for example, particlebombardment or Agrobacterium. See Example 1. Alternately, green tissuecan be made transgenic for a polynucleotide of interest by transformingan explant that can give rise to green tissue when the explant iscultured for a time and under conditions sufficient for the initiationand growth of green tissue to occur. As described, green tissueinduction is carried out under dim light. The length of exposure of theplant cells to dim conditions may vary based in part on the type ofplant species and genotype being transformed.

Any suitable explant that can give rise to green tissue may be used inthe methods described herein. The explant can be from a monocot. It willbe understood by one skilled in the art that the explant may comprise aplant cell, a tissue or an organ. Exemplary explants for use with themethods include but are not limited to embryos, green tissue, callussuch as Type I or II, cell suspensions, cotyledons, including scutella,meristems, seedlings, mature and immature seeds, leaves, stems, shoots,scutella, nodes, leaf bases, or roots. See U.S. patent applicationpublication no. 20080280361, U.S. Pat. Nos. 5,569,834; 5,416,011;5,824,877; 7,064,248. When the explant is an embryo from a maize plant,the method may include pollinating ears from the treated maize plant,harvesting the ears so that the ears or embryos may be prepared fortransformation. See, for example, Green and Phillips (Crop Sci.15:417-421, 1976). Maize immature embryos can be isolated frompollinated plants, as another example, using the methods of Neuffer etal. (“Growing Maize for genetic purposes.” In: Maize for BiologicalResearch W. F. Sheridan, Ed., University Press, University of NorthDakota, Grand Forks, N. Dak. 1982.). The explant may be prepared usingany suitable technique and may include, for example, isolating theexplant from the plant, excising plant cell, tissue, or organ from theexplant, sterilizing the plant cell, tissue, organ, or explant orcombinations thereof. In some cases, the explant is an embryo, such asan immature embryo from a monocot such as corn. In one example, themethods include transforming one or more immature embryos from themonocot using conventional methods such as Agrobacterium-mediatedtransformation or particle bombardment. See Example 1. As described,green tissue induction is carried out under dim light for a length oftime sufficient for the initiation and growth of green tissue to occur.The length of exposure of the plant cells to dim conditions may varybased in part on the type of plant species and genotype beingtransformed.

In cases where the explant is other than green tissue, the explant canbe used to generate green tissue using commonly known techniques. Forexample, green tissue can be obtained by culturing immature embryosunder appropriate conditions to initiate the formation of green tissue.See, for example, U.S. Pat. Nos. 6,541,257, 6,235,529, 7,102,056. WhenAgrobacterium is used to transform cells of the explant to generatetransgenic green tissue, typically the explant such as immature embryosare co-cultivated for about 1-3 days in the dark and rested for anadditional 1-3 days in resting medium, typically without selection. Theexplant is contacted with green tissue induction medium under dim lightto produce green tissue. The green tissue induction medium mayoptionally contain a selective agent, e.g. bialaphos and carbinicellin,when producing transgenic green tissue. Usually, the greentissue-induction media used in the methods contains differentcombinations of an auxin, cytokinin, and copper in amounts effective toinitiate the formation of green tissue. In one example of greentissue-induction medium the auxin is 2,4-dichlorophenoxyacetic acid(2,4-D), the cytokinin is 6-benzylaminopurine (BAP) and copper is CuSO₄.This culturing step usually takes about 2-3 weeks, preferably at about24° C.-28° C. under dim light.

In some circumstances, it may be desirable to break the green tissueinto one or more pieces to facilitate proliferation and more stringentselection. The method may also include subculturing the broken pieces ofgreen tissue in the presence of the selection agent for about 2-3 weeks.About 3 to 5 rounds of subculturing with a selective agent is typicallyconsidered sufficient to select for transformed tissue.

Transgenic green tissue can also be obtained by bombarding immatureembryos and culturing them under appropriate conditions to initiate theformation of green tissue. Immature embryos are isolated using anysuitable technique and placed scutellum-side up in an osmotic medium.The embryos are bombarded with solid particles, such as gold particles,coated with the polynucleotide of interest. The embryos are contactedwith green tissue induction medium that typically lacks a selectiveagent and cultured under dim light for about 3-7 days, usually at about24° C.-28° C. to produce transgenic green tissue. In some circumstances,it may be desirable to break the green tissue into one or more pieces tofacilitate proliferation and more stringent selection. The method mayalso include subculturing the broken pieces of green tissue in thepresence of the selection agent for about 2-3 weeks. About 3 to 5 roundsof subculturing with a selective agent is typically consideredsufficient to select for transformed tissue. Optionally, the putativetransgenic tissues are maintained and proliferated on green tissueinduction or maintenance medium containing a selective agent. Once asufficient amount of green tissues are obtained, the green tissue may beplated on solid regeneration/rooting medium optionally containing aselective agent and exposed to a higher light intensity, approximately45 to 100 μE m⁻²s⁻¹, on a 16-h light cycle. After about 4 to 6 weeks,regenerated plantlets may be transferred to soil.

When green tissues are used as targets for bombardment, the green tissuemay be pretreated with an osmotic solution. See Example 1. After about 4hours, the green tissue is bombarded using any suitable particle. Onetransformed, transgenic green tissues are selected and cultured in asimilar manner as that used for green tissue obtained by particlebombardment of immature embryos. See Example 1.

Transgenic regions of the green tissue obtained by any method may beconfirmed or identified using any suitable gene such as a maker gene.For convenience, visible marker genes such as RFP, GFP, EGFP,lucieferase or YFP are normally utilized to identify transgenic regionsin the green tissue using standard techniques and instruments such as afluorescence microscope. The transgenic regions of the green tissue maybe isolated by cutting the transgenic regions from the non-transgenicregions of the green tissue. The transgenic regions are typically cutinto several pieces and placed on maintenance medium for furtherproliferation. In some examples, the non-transgenic green tissue may becut into several pieces and placed on maintenance medium for furtherproliferation. In some cases, the maintenance medium and green tissueinitiation medium are the same.

The green tissues are transferred directly onto rooting medium so thatone or more roots are produced. Any suitable rooting medium may be used,including but not limited to phytohormone-free medium. For example, MSbasal medium supplemented with IBA (e.g., 0.5 mg/L) can be used toinduce root formation, if necessary. Depending upon the genotype,different levels of an auxin and cytokinin (i.e., a differentauxin/cytokinin ratio) provide optimal results. The medium may be of anysuitable form such as solid, liquid or gel, for example, medium gelledwith agar or an agar substitute gelling agent such as PHYTAGEL™(Sigma-Aldrich, St. Louis, Mo., USA). Normally, when the green tissue istransgenic the rooting medium contains a selective agent or one is addedto the medium. The green tissue is contacted with rooting medium thatinduces root formation for a time and under conditions sufficient toinitiate root growth from the green tissue, thereby producing aplantlet.

If desired, the green tissue may be incubated on regeneration mediumprior to placing tissues on rooting medium. Any suitable regenerationmedium can be used including without limitation to phytohormone-freemedium and others. One skilled in the art will be familiar with suchmedia. Exposing the green tissue to regeneration medium can facilitatemore efficient root production. The length of incubation is often for ashort period of time such as 1-3 weeks.

In one embodiment, the methods of the invention may use plant tissuesselected from, but not limited to, whole plantlets, plantlet parts,plantlet leaves or plantlet roots.

The following discussion is directed to assay of roots but can beadapted for the assay of other plant tissues including plantlets andleaves. Roots can be produced with shoot regeneration or without anyshoot regeneration. If shoot regeneration is desired, the green tissueis contacted with shoot regeneration medium for a sufficient length oftime and under conditions to generate shoots. In some instances, thegreen tissue may be contacted with shoot regeneration medium prior to,concurrent with, or subsequent to contacting the green tissue withrooting medium in order to produce a plantlet.

Plantlets having one or more roots may be removed from the solidifiedagar medium and the agar rinsed off the roots. In one aspect, the rootsare placed onto suitable assay dishes or containers such as a culturedish, e.g. Phyta trays or Petri dishes.

When doing so, it may prove advantageous to continue to keep the rootsgrowing and alive to more closely mimic real life infestations,infections or stresses of plants. Accordingly, the roots are placed in arooting medium such as MSA and MSB in the culture dish so that rootproduction is continuous. Typically, MSA includes MS salts and vitamins,2% sucrose, 0.35% Phytagel and 3 mg/L bialaphos and MSB includes MSsalts and vitamins, 2% sucrose, 0.25% Phytagel, 0.5 mg/L IBA and 3 mg/Lbialaphos. Additional exemplary rooting media are described above and inthe Examples herein. In some examples, the rooting medium lacks agar oran agar-substitute.

In another aspect, steps are taken to prevent or inhibit the growth ofunwanted contaminants e.g. microbes, such as bacteria, mold, or fungi inthe assay. For example, a biocide such as Plant Preservative Mixture(PPM-0.1350% 5-chloro-2-methyl-3(2H)-isothiazolone, 0.0412%2-methyl-3(2H)-isothiasolone, 99.8238% inert ingredients, Plant CellBiotechnology, Inc., Washington, D.C.) may be added to the rootingmedium to prevent or inhibit fungal and bacterial development.Additionally, the dish may be sterile or substantially sterile. Further,the use of sterile filter papers rather than agar in the dish can beused to facilitate the transfer of rooting solution to plant roots andreduce fungal and bacterial development. Any means, technique or object,such as filter paper, that facilitates the contact between the rootingmedium and roots may be used so long as it does not break off or killthe roots. Contact between the medium and the roots may be facilitatedby placing an object on top of the roots to force the roots downwardinto the dish. The object may be a screen or grid.

Use of the methods and assays described herein serve as an efficientmeans for testing endogenous genes or a polynucleotide of interest forvarious phenotypes in non-transgenic roots or roots transgenic for thepolynucleotide respectively. As described elsewhere herein, thepolynucleotide may be any suitable polynucleotide. Polynucleotidessuitable for use in the methods and assays described herein may beeither endogenous or heterologous to the plant cell of the explant to betransformed. The polynucleotide may be RNA, DNA or both. Polynucleotidesencompass all forms of nucleic acid sequences including, but not limitedto, single-stranded, double-stranded, triplexes, linear, circular,branched, hairpins, stem-loop structures, branched structures, and thelike. The polynucleotide may be a ds RNA molecule, such as a dsRNAmolecule that upon consumption by a pest decreases pest infestation. SeeU.S. Publication No. 20060021087. In some instances, the polynucleotideof interest may encode a polypeptide of interest which is alreadyexpressed in the native root cell. The polynucleotide of interest mayconfer or modulate one or more particular phenotypes of interest to theroot, for example, such as, but not limited to increased root size,increased overall root mass, altered root architecture, increasedexpression level of mRNA or protein, increased biochemical content,increased tolerance to stress, increased tolerance or resistance to apest, increased tolerance or resistance to a pathogen, increased yielddesirable agronomic traits, increased disease resistance, nutritionalenhancements, and the like. As will be appreciated by one skilled in theart, there may be overlap or correlations between the observedphenotypes.

Exemplary promoters to drive expression of the polynucleotide ofinterest include without limitation constitutive, inducible, orroot-preferred promoters and are described elsewhere herein and can beselected from the many available from the literature. Known or novelpromoters may be tested for functionality in a root cell, the relativestrength of the promoter in a root cell, the spatial expression of thepromoter in the root, or whether the promoter is a root-preferredpromoter using the methods and assays described herein.

The plant tissue is evaluated for expression levels of endogenouspolynucleotides of interest or heterologous polynucleotides of interest.Expression at the RNA level can be determined to identify and/orquantitate expression of a polynucleotide of interest. Standardtechniques for RNA analysis can be employed and include PCRamplification assays using oligonucleotides primers designed to amplifyonly the heterologous RNA templates and solution hybridization assaysusing heterologous nucleic acid-specific probes. The transgenic tissuemay be evaluated for the polynucleotide of interest's impact onresistance to diseases, pests, pathogens, stresses, nutrients, orchemicals and the like. The chemical may be a pesticide or bactericideand the like.

Roots may be evaluated for expression levels of endogenouspolynucleotides of interest or heterologous polynucleotides of interest,for example, in a transgenic root. Expression in the roots at the RNAlevel can be determined to identify and/or quantitative expression for apolynucleotide of interest. When the polynucleotide of interest is aroot-preferred promoter, as will be understood by one skilled in theart, the transgenic root may be evaluated for expression of thepolynucleotide operably linked to the root-preferred promoter. Standardtechniques for RNA analysis can be employed and include PCRamplification assays using oligonucleotides primers designed to amplifyonly the heterologous RNA templates and solution hybridization assaysusing heterologous nucleic acid-specific probes. If desired, the rootscan be analyzed for protein expression by fluorescent microscopy, FACS,or Western immunoblot analysis using the specifically reactive cognateantibodies. In addition, in situ hybridization and immunocytochemistryaccording to standard protocols can be done using heterologous nucleicacid specific polynucleotide probes and antibodies, respectively, tolocalize sites of expression within transgenic root tissue. Generally, anumber of transgenic roots are usually screened for the polynucleotideof interest to identify and select plantlets with the most appropriateexpression profiles, for example, in some examples, those thatcomparatively express the polynucleotide at the highest level or ascompared to a control null for the polynucleotide of interest. In somecases, it may be desirable to have low levels of expression of anendogenous gene in the root.

Roots, such as roots transgenic for the polynucleotide of interest, maybe evaluated for root size. Root size includes but is not limited toroot biomass, root strength, root thickness, the formation of aerialroots, the number of aerial roots, length of roots, and the number oflateral and/or adventitious roots and the like and combinations thereofas compared as to a control. See U.S. Pat. No. 7,259,296. In one aspect,expression of the polynucleotide of interest increases root biomass,produces thicker roots, produces stronger roots, increases the formationof aerial roots, increases the number of aerial roots, increases thelength of roots, and increases the number of lateral and/or adventitiousroots or combinations thereof.

In another aspect, the roots may be evaluated for the endogenous orheterologous polynucleotide of interest's impact on root architecture.Aspects of root architecture that may be evaluated include withoutlimitation root depth, root angle, root branching, number of root tips,nodal root diameter, nodal root volume, and root metabolic activity andthe like or combinations thereof. See U.S. Pat. No. 7,557,266. Oneskilled in the art will be familiar with techniques for determining suchaspects.

Expression of the endogenous or heterologous polynucleotide of interestmay also affect the biochemical content of the root. See, for example,J. Exp. Bot. (2003) 54: 203-211 describing the effect of pmt geneoverexpression on tropane alkaloid production in transformed rootcultures of Datura metel and Hyoscyamus muticus.

The transgenic roots may be evaluated for the polynucleotide ofinterest's impact on resistance to diseases, pests, pathogens, stresses,nutrients, or chemicals and the like. The chemical may be a pesticide orbactericide and the like.

Accordingly, the embodiments encompass methods that are directed toprotecting plants against root pathogens or biotic stresses such asfungal pathogens, bacteria, viruses, nematodes, pests, and the like. By“disease resistance” or “insect resistance” is intended that the plantsavoid the harmful symptoms that are the outcome of the plant-pathogeninteractions. U.S. Pat. No. 7,456,334. Pathogens of the embodimentsinclude, but are not limited to, viruses or viroids, bacteria, insects,nematodes, fungi, and the like. Viruses include tobacco or cucumbermosaic virus, ringspot virus, necrosis virus, maize dwarf mosaic virus,etc. Nematodes include parasitic nematodes such as root knot, cyst, andlesion nematodes, etc. As described elsewhere herein, various changes inphenotype may be determined in the plantlet root, e.g. altering aplant's pathogen or insect defense mechanism or increasing the plant'stolerance to herbicides

Advantageously, the present methods and assays use an intact, live rootthat takes place in a dish and allows for the continuous generation ofplants from roots obtained from the green tissue. With respect to thecontinuous generation of transgenic plants from transgenic green tissue,advantageously these may be obtained from the same transgenic event.This is in contrast to other rootworm bioassay techniques that employground-up transformed roots or use seedlings in soil which are infestedwith either eggs or neonates. The former destroys the plant and requiresnew plants to be recreated with the same genomic character. The latterrequires an assay of at least 14 days of duration and is oftendestructive in nature. It is challenging to determine the activity ofthe plant on the larvae as it is difficult to find the larvae in thesoil. Advantageously, the status of the pests and root are easilyobservable using the assays and methods described herein since they donot require that the roots be immersed or buried in soil.

The assays and methods described herein are also economical from a timeand space standpoint as they have a duration of about 1 to 14 days orless and extensive greenhouse space is not needed to perform the methodsand assays, rather they can be performed in an incubator with lights. Inone embodiment of the invention, the effect of insect application onplantlet, root or leaf damage may be assayed within about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or more days after infestation with oneor more pests.

Pests that may be used in the methods and assays include withoutlimitation those insects belonging to the order of Lepidoptera, whichwould feed on the stalk and leaves, e.g. European corn borer (Ostrinianubilalis), Corn earworm (Helicoverpa zea), Fall armyworm (Spodopterafrugiperda), Western bean cutworm (Richia albicosta), Black cutworm(Agrotis ipsilon), Lesser cornstalk borer (Elasmopalpus lignosellus),Southwest corn borer (Diatraea grandiosella), Sugarcane borer (Diatraeasaccharalis), Homoptera, e.g. Aphid (leaf feeding and root feeding),Leafhoppers, Coleoptera, e.g. Corn rootworms, Wireworms, or White grubs(Scarabs) or Heteroptera. In another aspect, steps are taken to preventor inhibit the growth of unwanted contaminants e.g. microbes, such asbacteria, mold, or fungi in the assay. For example, a biocide such asPPM may be added to the medium to prevent or inhibit fungal andbacterial development. Further, the use of an absorbent material such asfilter papers, rather than agar, in the Phytatray or Petri dish can beused to facilitate the transfer of nutrient solution to plant roots,while reducing fungal and bacterial development. If further contact isdesired between the medium and the roots, a wire grid or screen may beplaced on top of the roots.

Pests may be sterilized prior to contact with the root. In someexamples, the eggs, larvae, instars or adults of the pests are treatedto remove or kill bacterial or fungal spores which may include washingwith once or multiple times with a solution such as ethanol or CHLOROX®bleach (The Chlorox company, Oakland, Calif.). The pests may be feedprior to infesting the roots. For example, neonates may be placed onartificial diet for 24 hours prior to being placed on the test roots.This eliminates any larvae that will have died in the initial 24 hoursas well as allowing for the selection of uniform-sized and healthy testsubjects. Allowing the neonates to feed for 24 hours provides thefurther benefit of causing the evacuation of any fungal or bacterialspores in the gut with the elimination of frass. In another aspect, thepest in any developmental stage such as eggs, larvae, instars, or adultsmay be sprayed with LYSOL® disinfectant, e.g. EPA Reg No 777-72, inparticular Professional LYSOL® disinfectant spray, EPA Reg No777-72-625, or another disinfectant prior to infestation to help killfungi and bacteria (LYSOL® disinfectant Reckitt Benckiser Inc,Parsippany, N.J.).

Subsequent to pest infestation or exposure to the stress or pathogen,the root is incubated under appropriate conditions, for example,incubating the dish plus roots and pests at about 24° C.-28° C. underlight or dark conditions. The roots may be subjected to pests of theappropriate developmental stage, for example, larvae, and appropriatenumber. Generally the duration of the assay is about 4-14 days. Thephenotypes of the roots or pests or both may be observed at any suitabletime point but are typically performed at completion. As understood byone skilled in the art, the damage to roots and pests can be determinedin various ways, including objective and subjective techniques. Forexample, the roots may be scored for their damage by the pests on ascale of 0 to 5 with 0 indicating little or no observable damage tosevere root damage. In addition, leaf damage can also be scored fordirect feeding by the insects, or by color changes and wilting due todamage to the roots or stem. In some instances, color change in variousplantlet parts, such as the leaves, stems, and/or roots, may be observedas a result of pest damage. Color change may occur in none, some or allof these parts. Pests may be scored to count “live” versus “dead” or“stunted” larvae and tabulating the results to express as a percentageof mortality. Any result of dead or stunted or combinations thereof over50% is considered a positive result. In another aspect, the roots may beevaluated for resistance to any rootworm, for example, resistance toSouthern corn rootworm (Diabrotica undecimpuncata), Western cornrootworm (Diabrotica virgifera), and/or Northern corn rootworm(Diabrotica barber), and the like. In some cases, the transgenic rootsmay be evaluated for the polynucleotide of interest's impact onresistance to a pest, such as any rootworm.

As another example, the plantlet may be scored for damage by the pestsby observing the color change of the stem area above the crown of theroots. With respect to a normal plantlet of maize exposed to a pest,when the stem area above the crown darkens from pest damage, e.g. fromWCRW damage; this color change indicates that the polynucleotide ofinterest is not effective for controlling pest infestation and/or damageto the stem. However, when little or no color change of the stem areaabove the crown is observed when the plantlet is exposed to a pest, thisindicates that the polynucleotide of interest is effective forcontrolling pest infestations and/or damage to the plantlet. As anotherexample, the crown of the plantlet may be scored for damage by the pestsby determining the existence of holes in the crown. The number of holesin the crown can be translated into a numerical value which can be usedto determine the overall activity of the polynucleotide of interest inprotecting the plantlet.

Transgenic plants may be regenerated from green tissue that has a roottesting positive for a desirable phenotype. A plant having the desiredphenotype may be produced by regenerating the plant from the greentissue and the resultant plant entered into a plant breeding program.After 3-4 weeks, the regenerated transgenic plantlets may be transferredto soil and grown into a transgenic plant in a greenhouse. Accordingly,in one aspect, the methods may include growing the transgenic plantletinto a transgenic plant. Transgenic seed may also be obtained from theplant if desired.

Any well-known regeneration medium may be used for the practice of theprovided methods. “Regeneration medium” (RM) promotes differentiation oftotipotent plant tissues into shoots, roots, and other organizedstructures and eventually into plantlets that can be transferred tosoil. Auxin levels in regeneration medium are reduced relative to MPMor, preferably, auxins are eliminated. It is also preferable that copperlevels are reduced, e.g., to levels common in basal plant culture mediasuch as MS medium. It is preferable to include a cytokinin in RM, ascytokinins have been found to promote regenerability of the transformedtissue. However, regeneration can occur without a cytokinin in themedium. Typically, cytokinin levels in RM are from about 0 mg/L to about4 mg/L. RM also preferably includes a carbon source, preferably about 20g/L to about 30 g/L, e.g., either sucrose or maltose.

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerwhich has been introduced together with a polynucleotide of interest.For transformation and regeneration of maize see, Gordon-Kamm, et al.,(1990) The Plant Cell 2:603-618.

Regeneration can also be obtained from explants, green tissue, roots,plantlets, or parts thereof. Such regeneration techniques are describedgenerally in Klee, et al., (1987) Ann. Rev. of Plant Phys. 38:467-486.The regeneration of plants from either single plant protoplasts orvarious explants is well known in the art. See, for example, Methods forPlant Molecular Biology, Weissbach and Weissbach, eds., Academic Press,Inc., San Diego, Calif. (1988). This regeneration and growth processincludes the steps of selection of transformant cells and shoots,rooting the transformant shoots and growth of the plantlets in soil. Formaize cell culture and regeneration see generally, The Maize Handbook,Freeling and Walbot, Eds., Springer, New York (1994); Corn and CornImprovement, 3.sup.rd edition, Sprague and Dudley Eds., American Societyof Agronomy, Madison, Wis. (1988).

Plants to be transferred to the growth chamber are removed from sterilecontainers and the solidified agar medium is rinsed off the roots. Theplantlets are placed in a commercial potting mix in a growth chamberequipped with a misting device which maintains the relative humiditynear 100% without excessively wetting the plant roots. Approximatelythree to four weeks are required in the misting chamber before theplants are robust enough for transplantation into pots or into fieldconditions. At this point, many plantlets, especially those regeneratedfrom short-term callus cultures will grow at a rate and to a sizesimilar to seed-derived plants. Plants regenerated from long-termcallus, from suspension cultures, and from in vitro-selected callus willsometimes show phenotypic abnormalities, such as reduced plant size,leaf striping and delayed maturation. Care must be taken to assurecontrolled pollination with such plants. Ten to fourteen days afterpollination, the plants are checked for seed set. If there is seed, theplants are then placed in a holding area in the greenhouse to mature anddry down. Harvesting is typically performed six to eight weeks afterpollination.

One of skill will recognize that after the recombinant expressioncassette comprising the polynucleotide of interest is stablyincorporated in transgenic plants and confirmed to be operable, it canbe introduced into other plants by sexual crossing. Any of a number ofstandard breeding techniques can be used, depending upon the species tobe crossed.

This invention can be better understood by reference to the followingnon-limiting examples. It will be appreciated by those skilled in theart that other embodiments of the invention may be practiced withoutdeparting from the spirit and the scope of the invention as hereindisclosed and claimed.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. The disclosure of each reference set forthherein is incorporated herein by reference in its entirety.

Example 1 Production of Transgenic Maize Events Via Bombardment ImmatureEmbryos as a Bombardment Target

Ears of a maize (Zea mays L.) cultivar, PHR03, were surface-sterilizedfor 15-20 min in 20% (v/v) bleach (5.25% sodium hypochlorite) plus 1drop of Tween 20 followed by 3 washes in sterile water. Immature embryos(IEs), typically 9 to 12 days after pollination, were isolated from earsand were placed scutellum-side up in an osmotic medium containingequimolar amounts of mannitol and sorbitol to give a final concentrationof 0.4 M. The embryos were bombarded with gold particles coated with DNAcontaining bar/moPAT or another selectable marker using a PDS-1000 Hebiolistic device (Bio-Rad, Inc., Hercules, Calif.) at 650-1300 psi.Between 16 hr and 18 hr after bombardment, the bombarded embryos wereplaced on green tissue induction medium without osmoticum and grown at26° C.±2° C. under dim light (10-50 uE m⁻² s⁻¹). Following the initial 4to 10 day culturing period, each green tissue was broken into 1 to 3pieces depending on tissue size and transferred to green tissueinduction medium supplemented with bialaphos or another selective agent.Three weeks after the first round of selection, cultures weretransferred to fresh green tissue induction medium containing aselective agent at 3 to 4 week intervals. Following identification ofsufficient sized green, regenerative structures, tissues were thentransferred directly onto 2 different shoot and root regenerationculturing schemes: (1) 7-14 days of incubation on 289F shootregeneration medium prior to placing tissues on rooting medium and (2)directly onto rooting medium. Two rooting media were also tested: (1)MSA containing MS salts and vitamins, 2% sucrose, 0.35% Phytagel and 3mg/L bialaphos and (2) MSB containing MS salts and vitamins, 2% sucrose,0.25% Phytagel, 0.5 mg/L IBA and 3 mg/L bialaphos. MSB was moreefficient in root formation than MSA.

Green Tissues as a Bombardment Target

Ears of PHR03 were surface-sterilized as described above. Green tissueswere induced and proliferated by culturing IEs on green tissue inductionmedium and used for bombardment. Green tissues, approximately two tothree months old, were used as targets for bombardment. Tissues (4 to 6mm) were transferred for osmotic pretreatment to green tissue inductionmedium containing 0.2 M mannitol and 0.2 M sorbitol. After 4 hr, tissueswere bombarded as described above. Sixteen to 18 hr after bombardment,the bombarded tissues were placed on green tissue induction mediumwithout osmoticum and grown at 26° C.±2° C. under dim light (10-50 uEm⁻² s⁻¹). Following the initial 4 to 10 day culturing period, each greentissue was broken into 1 to 3 pieces depending on tissue size andtransferred to green tissue induction medium supplemented with bialaphosor another selective agent. Three weeks after the first round ofselection, cultures were transferred to fresh green tissue inductionmedium containing a selective agent at 3 to 4 week intervals. Oncetransformed, transgenic green tissues are selected and cultured in asimilar manner as that used for green tissue obtained by particlebombardment of immature embryos.

Example 2 Production of Transgenic Maize Events Via AgrobacteriumPreparation of Agrobacterium Suspension:

Agrobacterium tumefaciens harboring a binary vector containing DS-RED(RFP) reporter gene and a selectable marker (moPA 7) with or without aBt gene was streaked out from a −80° frozen aliquot onto solid PHI-Lmedium and cultured at 28° C. in the dark for 2-3 days. PHI-L mediacomprised 25 ml/L stock solution A, 25 ml/L stock solution B, 450.9 ml/Lstock solution C and spectinomycin added to a concentration of 50 mg/Lin sterile ddH₂O (stock solution A: K₂HPO₄ 60.0 g/L, NaH₂PO₄ 20.0 g/L,adjust pH to 7.0 with KOH and autoclave; stock solution B: NH₄Cl 20.0g/L, MgSO₄.7H₂O 6.0 g/L, KCl 3.0 g/L, CaCl₂ 0.20 g/L, FeSO₄.7H₂O 50.0mg/L, autoclave; stock solution C: glucose 5.56 g/L, agar 16.67 g/L andautoclave). Two ways to grow Agrobacterium were used for transformation.

1. Growing Agrobacterium on Solid Medium

A single colony or multiple colonies were picked from the master plateand streaked onto a plate containing PHI-M medium and incubated at 28°C. in the dark for 1-2 days.

Five mL Agrobacterium infection medium and 5 μL of 100 mM3′-5′-Dimethoxy-4′-hydroxyacetophenone (acetosyringone) were added to a14 mL Falcon tube in a hood. About 3 full loops of Agrobacterium weresuspended in the tube and the tube was then vortexed to make an evensuspension. One mL of the suspension was transferred to aspectrophotometer tube and the OD of the suspension was adjusted to 0.35at 550 nm. The Agrobacterium concentration was approximately 0.5×10⁹cfu/mL. The final Agrobacterium suspension was aliquoted into 2 mLmicrocentrifuge tubes, each containing 1 mL of the suspension. Thesuspensions were then used as soon as possible.

2. Growing Agrobacterium on Liquid Medium

One day before infection, a 125 mL flask was set up with 30 mLs of 557Aand 30 uL spectinomycin (50 mg/mL) and 30 uL acetosyringone (20 mg/mL).A half loopful of Agrobacterium was suspended into the flasks and placedon a 200 rpm shaker at 28° C. overnight. The Agrobacterium culture wascentrifuged at 5000 rpm for 10 min. The supernatant was removed and theAgrobacterium infection medium+acetosyringone solution was added. Thebacteria were resuspended by vortex and the OD of Agrobacteriumsuspension was adjusted to 0.35 at 550 nm.

Maize Transformation:

Ears of maize (Zea mays L.) cultivars, PHR03 and PH4CN, weresurface-sterilized for 15-20 min in 20% (v/v) bleach (5.25% sodiumhypochlorite) plus 1 drop of Tween 20 followed by 3 washes in sterilewater. Immature embryos (IEs) were isolated from ears and were placed in2 mL of the Agrobacterium infection medium plus acetosyringone solution.The optimal size of the embryos was 1.5-1.8 mm and 1.3-2.1 mm for PHR03and PH4CN, respectively. The solution was drawn off and 1 mL ofAgrobacterium suspension was added to the embryos and the tube vortexedfor 5-10 sec.

The microfuge tube was allowed to stand for 5 min in the hood. Thesuspension of Agrobacterium and embryos were poured onto co-cultivationmedium. Any embryos left in the tube were transferred to the plate usinga sterile spatula. The Agrobacterium suspension was drawn off and theembryos placed axis side down on the media. The plate was sealed withParafilm tape and incubated in the dark at 21° C. for 1-3 days ofco-cultivation.

Embryos were transferred to resting medium without selection. Three to 7days later, they were transferred to green tissue induction mediumcontaining 3-5 mg/L bialaphos (Meiji Seika K.K., Tokyo, Japan) plus 100mg/L carbenicillin (ICN, Costa Mesa, Calif.). The plate was sealed withParafilm and incubated at 26° C.±2° C. under dim light. At 2-3 weeksafter the first round selection, each callusing piece, broken into 1 to3 pieces, depending on initial size, was transferred to fresh mediumsupplemented with a selective agent. Tissues were subcultured on freshmedium containing bialaphos at 2-3 week intervals. At 3^(rd) roundselection, transgenic sectors were identified by visible markers (e.g.RFP) under a fluorescence microscope and chopped into small pieces toplace on maintenance medium for further proliferation. Transgenic greentissues were proliferated until sufficient amount of tissues wasobtained. Table 1 below shows transgenic events produced from PHR03 andPH4CN using different Bt gene constructs.

TABLE 1 Transgenic maize events transformed with different Bt geneconstructs #transgenic Construct Inbred Bt gene events/ PHP26650 PH4CNControl 37 PHR03 44 PHP36779 PH4CN Shuffled Bt variant 12 2A12-V1 PHR032 PHP36782 PH4CN Shuffled Bt variant 14 2A12-V2 PHR03 38 34651- Bt PH4CNBt variant V6 2 Variant V6 PHR03 169 34651- Shuffled Bt PH4CN ShuffledBt variant 3 variant 2A12-V5 PHR03 2A12-V5 86 34651- Shuffled Bt PH4CNShuffled Bt variant — variant 2A12-V3 PHR03 2A12-V3 48 34651- ShuffledBt PH4CN Shuffled Bt variant — variant 2A12-V4 PHR03 2A12-V4 49

Example 3 Continuous Root or Root/Shoot Production from GreenRegenerative Tissues of Transgenic Maize Events

Highly regenerative, green tissues of monocot crops species containmultiple, light to dark green, shoot meristem-like structures. Thesetissues regenerate multiple shoots without loss or with minimum loss ofregenerability for more than a year. These green tissues areorganogenic, rather than embryogenic, and are likely to havemeristem-like tissues which are ready for shoot regeneration and rootformation. Transgenic sectors were identified by visible markers under afluorescence microscope and chopped into small pieces to place onmaintenance medium for minimal proliferation. Tissues were thentransferred directly onto 2 different shoot and root regenerationculturing schemes: (1) 7-14 days of incubation on 289F shootregeneration medium prior to placing tissues on rooting medium and (2)directly onto 2 rooting media, MSA and MSB. MSB was more efficient inroot formation than MSA. When green tissues were incubated directly onMSB, both shoot and root formation or root formation only was observed.The use of 289F shoot regeneration medium could facilitate shootproduction more efficiently. Transgenic roots could be produced withoutany shoot regeneration when green tissues with no shoot regeneration on289F were transferred onto MSB Regenerated shoot and root tissues showeduniform expression of RFP. This system can be used to do quick genetesting in roots such as corn root worm assay and functionality test ofroot-specific promoters using stably transformed tissues.

Example 4 Screening of Transgenic Maize Events with Bt Gene Expressionby Western Blot Analysis

In order to screen transgenic events with Bt gene expression, westernblot hybridization analysis was carried out. Forty to 100 mg of greentissues or leaf or root tissues from each transgenic event were mixedwith 0.1 to 0.25 mL CCLR protein extraction buffer (100 mM phosphatebuffer, 1 mM EDTA, 1% Triton, 10% Glycerol, 7 mM BME, pH 7.8) (Cat.#E1531, Promega Corp., Madison, Wis.) in a 2 mL microfuge tube. Afteradding two steel balls in the tube, the samples were ground two to threetimes with the GenoGrinder2000 (1× rate, 2 min 30 sec/run; 250strokes/min). After centrifugation (10,000×g for 2 min), 30 μL of thesupernatant and 10 μL of 4× loading dye were mixed in a fresh tube andheated for 5 min at 95° C. Twenty μL of total soluble protein (total6000 μg wet tissue equivalent) from each event and purified Bt proteinas a positive control were separated on SDS-PAGE using NuPAGE 4-12% BisTris gel (Invitrogen Corp, Carlsbad, Calif.) and transferred tonitrocellulose membrane. After transfer, the membrane was blocked inTBS-T (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20)+5% nonfatdried milk overnight. After washing (2×5 min) in TBS-T, primary rabbitpolyclonal Bt antibody was added at 1:1000 dilution and incubated for1.5 to 2 hr at room temperature. After washing in 1% BSA/TBS-T, themembrane was incubated in goat anti-rabbit alkaline phosphatase (AP)antibody at 1:1000 dilution for 1-2 hr at room temperature and washed asindicated above. Labeling was monitored by using Bio-Rad (Hercules,Calif.) AP conjugate substrate kit for detection according tomanufacturer's instructions. Expression signal was quantified usingUN-SCAN-IT gel (Gel & Graph Digitizing Software 6.1, Silk Scientific,Inc., Orem, Utah).

Bt expression in transgenic events was determined by western analysisusing green tissues and leaf and root tissues from each event. Out of 6Bt variants, shuffled Bt variant 2A12-V5 had the highest expression ofBt (60 ppm) in green tissues while shuffled Bt variant 2A12-V2 waslowest in expression (7 ppm). Shuffled Bt variants 2A12-V1, 2A12-V3,2A12-V4, and 2A12-V6 showed 26, 36, 28 and 38 ppm, respectively. Ingeneral, root and leaf tissues had much lower levels of Bt expression,compared with that of green tissues derived from the same event, e.g.,root (7 ppm) and leaf tissues (6 ppm) vs. green tissues (23 ppm) fromshuffled Bt variant 2A12-V4 event #2. For each construct 3 to 8 highestexpressors were selected for shoot and/or root regeneration for Westerncorn rootworm (WCRW) bioassay.

Example 5 Establishment and Optimization of a Contamination-Free InVitro Insect Bioassay System Using Live Plantlets or Root TissuesPreparation of Plantlets or Root Tissues:

Plantlets used for western corn rootworm bioassay were selected fromhigh expression events containing Bt genes. Plantlet selections werebased on root thickness and root mass, preferably with a single shootand either thin young roots or highly branched young roots. A mixture ofMSA with 0, 1, 1.5, 2 and 5% Plant Preservative Mixture (PPM—0.1350%5-chloro-2-methyl-3(2H)-isothiazolone, 0.0412%2-methyl-3(2H)-isothiasolone, 99.8238% inert ingredients, Plant CellBiotechnology, Inc.) was prepared for plantlet soak and as a nutrientsolution. Two mL MSA-PPM mixture on two 70 mm circular Whatman(Schleicher & Schuell) filter papers in Petri dishes (100×15 mm, VWR)were set-up for plantlet/root containment. Individual plantlets wereextracted from MSB media and roots were removed from agar by forceps.Extraneous yellow and browning tissue, leaves and roots were cut away orexcised. Each plantlet was soaked in 50 mL MSA containing differentlevels of PPM for 3 to 5 minutes. After soaking, stray residual agar andextra nutrient solution were removed from each plantlet, and the rootsthen arranged for maximum contact with the filter paper within theprepared Petri dishes, one plantlet per Petri dish. Plant materials weresubmitted for 1 day old WCRW infestation Root tissues alone withoutleaves also could be prepared for the assay using the same protocoldescribed above.

Preparation of Western Corn Root Worms:

Non-diapausing WCRW eggs were cleaned and surface sterilized insuccessive steps using a modification of the procedure described byMarrone et al. (1985) (J of Economic Entomology 78: 290-293), with 1%Clorox bleach, 0.25% peracetic acid solution, 70% ethanol, and thende-ionized water. The final step utilizes washing with deionized waterfour times. Eggs were suspended in a 0.22% agarose solution andtransferred to agar Petri plates covered with two 9 cm filter paperdisks. Once dry, the eggs were sprayed with a thin layer of ProfessionalLysol Disinfectant Spray (28.1% a.i.). After they were dry, the plateswere covered and four dishes were placed in 32 ounce Nalgene containersthat had been sprayed with Lysol, wiped down, and allowed to dry. TheNalgene containers were closed and put in incubators kept at 28° C. inthe dark for two days until egg hatch. Neonates were transferred tosingle-well plates with artificial WCRW diet, sealed with heat-sealableMylar with pin holes, and kept at 25° C. in the dark for 24 hr.

Infestation of Plantlets with Western Corn Root Worms:

Under a laminar flow hood, larvae were tapped into a heat-sterilized #80sieve. Larvae were sprayed with or without Professional LysolDisinfectant Spray (28.10% a.i.) and allowed to soak for 2 min. Thelarvae were then washed with autoclaved water and transferred to a 50 mLcentrifuge tube. Additional autoclaved water was added to the tube andthe larvae swirled for 1 minute. The water was decanted, leaving thelarvae and a small amount of water in the bottom of the tube. A steriledisposable inoculating loop was used to transfer ˜25 larvae to the Petridishes containing the plantlet. The Petri dish/plantlet/larvae areplaced in an incubator at 25° C. in a 16-hr light phase for 5-7 daysprior to checking contamination of the plates and plants.

No fungal or bacterial contamination was observed in the plates with MSAsolution plus 5% PPM when non-sterilized rootworms were infested (Table2). In contrast, fungal contamination was detected with 1% or 2% PPM;the higher the level of PPM, the less the contamination. Rootworms,however, were severely stunted at 5% PPM.

TABLE 2 Number of contaminated plates treated with non-sterilized WCRWsat different PPM concentrations in liquid MSA medium. PPM level 0% PPM1% PPM 2% PPM 5% PPM # plates 2/2 1/2 1/2 0/2 contaminated/# (severemold (severe mold (minor mold (No mold) plates tested contamination)contamination) contamination) The MSA-PPM mixtures on 2 Whatman filterpapers in Petri dishes were set for plantlet/root containment.

Another set of experiments was conducted with rootworms sterilized withLysol. Fungal contamination was controlled in the plates with both 1%and 1.5% PPM treatments (Table 3), but mild bacterial contamination wasoccasionally observed at 1% PPM. When sterilized rootworms were placedonto agar medium, severe bacterial contamination was detected (data notshown). Based on these results, a filter paper system supplemented withMSA liquid medium containing 1.5% PPM was employed using rootwormssterilized with Lysol for further WCRW bioassay.

TABLE 3 Number of contaminated plates treated with sterilized WCRWs 1%PPM 1.5% PPM Non-sterilized 1/2 1/2 rootworm (severe mold (minor moldcontamination contamination Sterilized 0/2 0/2 Rootworm w/Lysol (NoContamination) (No Contamination)

Example 6 Western Corn Rootworm Bioassay Using Transgenic Maize EventsExpressing Bt Genes

Transgenic plant materials were produced as described in Example 1-3.Plantlets/root tissues were prepared as described in Example 3. One to 3plantlets per event were extracted from MSB media and roots were removedof agar by forceps. After soaking in MSA containing 1.5% PPM, strayresidual agar and extra nutrient solution were removed from eachplantlet, and the roots were then arranged for maximum contact with 3filter papers within the prepared Sigma Phytatrays, one plantlet perPhyatray vessel. Roots not in contact with the filter paper were weigheddown by single 2″×2″ stainless steel mesh screens until it wasaccessible to the nutrient solution. About 20 plantlets were preparedfor WCRW bioassay, as well as 1 to 3 control plantlets per inbred line,and used for WCRW infestation.

Under a laminar flow hood, the one-day-old larvae were transferred to aheat-sterilized #80 sieve and sprayed until drenched with ProfessionalLysol Disinfectant Spray and allowed to soak for 2 min. The larvae werethen washed with autoclaved water and transferred to a 50 mL centrifugetube and swirled for 1 min. The water was decanted, leaving the larvaein a small amount of water in the bottom of the tube. A steriledisposable inoculating loop was used to transfer ˜25 larvae to thePhytatray containing the transformed plantlet. After infestation, thePhytatrays were then placed in an incubator at 25° C. in a 16-hr lightphase for 7 days prior to scoring the plants and larvae for plant damageand larval development.

As shown, 24 to 36% of larvae developed to the 2^(nd) instars (Table 4)and root tissues had severe damage on control plantlets of both PH4CNand PHR03 1 wk after infestation while the growth of rootworms wasseverely stunted and little root damage was detected on Herculex®plantlets (Table 4). Herculex® RW contains Cry 34/Cry35 toxins. (Pioneerand Dow Agro). The expression level of 6 Bt variants was 7 to 60 ppm ingreen tissue. In general the higher the Bt expression of the same Btvariant in green tissue, the more resistant the plantlets and roots torootworms (data not shown). A few events with high Bt expression ingreen tissue were susceptible to rootworms; these events did not show Btexpression in roots possibly due to production of multiple eventsderived from the same embryo or transgene silencing as tissue reachesstages of plantlets or roots. All Bt variants tested, except variant2A12-V2, were slightly to moderately resistant to rootworms. (Table 4).Event #3 transformed with Bt variant V6 (at 21 ppm) had little rootdamage and stunted root worms 4 days after infestation (data not shown).Event #23 transformed with shuffled variant 2A12-V3 (at 9 ppm) hadsevere root damage and bigger root worms 4 days after infestation. (datanot shown). Event #23 transformed with variant 2A12-V3 (Table 4) and 3events (#s 3, 5 and 7) with variant 2A12-V2 were low in efficacy,possibly due to low expression (Table 4). The number of the 2^(nd)instars and root damage appeared to be the best indicators to evaluatethe efficacy of Bt genes and transgenic events (Table 4).

TABLE 4 WCRW bioassay results using T₀ PH4CN and PHR03 plantlets CrownRoot Leaf Expt Inbred Construct Event # Rep # II instars Damage DamageDamage Plant Bt expression in tissue, ppm #1 PH4CN 26650 6 0 #1 6 ++ ++none live (−control) Shuffled 5 12 #1 2 + + none live Bt variant 11 12#1 2 none + none live 2A12-V1 Shuffled 3 7 #1 4 ++ ++ none live Btvariant 5 6 #1 5 +++ ++ none live 2A12-V2 7 5 #1 8 +++ +++ none liveHerculex ® (+control) n.a. #1 0 none (+) none live RW Bt expression inGT, ppm #2 PHR03 26650 6 0 #1 9 +++ +++ some dying (−control) 0 #2 6 ++++++ moderate dead Shuffled 22 27 #1 3 + ++ none healthy Bt variant 27 #21 + ++ some dying 2A12-V3 23 9 #1 12 +++ +++ some dying Shuffled 3 21 #10 none + none dying Bt variant 21 #2 0 + ++ none dying 2A12-V6 21 #3 0none none none healthy 10 23 #1 7 ++ ++ some dying 23 #2 0 ++ + nonehealthy Herculex ® (+control) n.a. #1 0 none + some healthy RW

Example 7 Application of the Current WCRW Bioassay System to OtherInsects

This example illustrates significant pest inhibition obtained by feedinglepidopteran larvae on corn tissue transformed with Bt genes.

Corn earworm, fall armyworm, black cutworm and sugarcane borer eggs werereceived from Benzon Research (Carlisle, Pa.). European corn borer eggswere received from Pioneer (Johnston, Iowa). Soybean looper eggs werereceived from DuPont (Wilmington Experiment Station, DE). Eggs were keptat 28° C. and allowed to hatch. Neonates were placed on a multi-specieslepidopteran diet (Southland Products, Lake Village, Ak.) and kept at28° C. for 24 hr. Under a laminar flow hood, 10 one-day-old larvae weretransferred to the Phytatray containing the transgenic plantlet using asterile disposable inoculating loop. Prior to infestation, a minimalamount of plant nutrients and 1.5% PPM was applied to the 3 sterilefilter papers on which the plantlet was placed, as described in Examples4, 5 and 6. Metal mesh screen was placed on the roots of the plantlet toinsure good contact between the roots and the plant nutrients on thefilter paper if necessary. The Phytatrays were then placed in anincubator at 26° C. in a 16-hr light phase for 3-7 days prior to scoringthe plants and larvae for plant damage and larval development. Plantswere observed to determine those expressing Bt to kill or stunt thelepidopteran pests compared to the control which were not expressingtoxin.

Table 5 demonstrates the effect of Bt 1 expression in corn plantlets onresistance to soybean looper. All transgenic events (#s 2, 16, 17, and33) showing the Bt1 gene presence had good resistance to soybean looperwhile negative control (#583) and Herculex®RW without Bt1 weresusceptible to soybean looper (Table 5)

TABLE 5 In vitro soybean looper bioassay results using T₀ PHR03plantlets Construct Bt1 gene Insect # Insect # Condition of leaves(event #) presence alive dead (0% to 100%) Condition of insects 24600(#583) − 20 0 50% eaten Several third instars Bt1 (#40) − 7 13 20% eatenAll seven still 1st instars (stunted) Bt1 (#33) + 0 20 0% eaten All deadneonates Bt1 (#17) + 0 20 0% eaten All dead neonates Bt1 (#16) + 0 20 0%eaten All dead neonates Bt1 (#2) + 0 20 2% eaten, slight All deadneonates holes in leaves Herculex ®RW − 14 6 80% eaten Various instarsup to 3rds Twenty neonates of soybean looper were infested onto cornplantlets. Five days after infestation plant damage and insect growth,stunting and survival were scored.

Table 6 demonstrates the effect of Bt 1 expression in corn plantlets onresistance to black cutworm. Transgenic event #54 showing the Bt1 genepresence had good resistance to black cutworm while event #40 withoutBt1 was susceptible to black cutworm.

TABLE 6 In vitro black cutworm bioassay results using T₀ PHR03 plantletsBt1 gene Insect # Insect # Plant presence alive dead Leaf damage healthBt1 (#54) + 3 7 10%, stem & leaves pocked Alive Bt1 (#40) − 10 0 95%,stem completely shredded, Dead everything eaten Herculex ®RW − 5 5 80%,stem shredded; leaves Dead shredded Ten neonates of black cutworm wereinfested onto corn plantlets. Seven days after infestation plant damageand insect growth, stunting and survival were scored.

Table 7 demonstrates the effect of Bt 1 expression in corn plantlets onresistance to corn earworm. Transgenic event #s 25, 42 and 82 showingthe Bt1 gene presence had good resistance to corn earworm while event#40 and Herculex®RW without Bt1 were susceptible to corn earworm.

TABLE 7 In vitro corn earworm bioassay results using T₀ PHR03 plantletsBt1 gene Insect # Insect # presence alive dead Leaf damage Plant healthBt1 (#42) + 0 10 0%, no penetration Healthy Bt1 (#82) + 0 10 0%, nopenetration Healthy Bt1 (#25) + 0 10 0%, one penetration hole, Healthybut did not go anywhere Bt1 (#40) − 10 0 95%, stem completely Deadshredded, everything eaten Herculex ®RW − 5 5 80%, stem shredded; leavesDead shredded Ten neonates of corn earworm were infested onto cornplantlets. Seven days after infestation plant damage and insect growth,stunting and survival were scored.

Table 8 demonstrates the effect of Bt 1 expression in corn plantlets onresistance to fall armyworm and sugarcane borer. All transgenic eventsexcept event #20 showing the Bt1 gene presence had good resistance toboth fall armyworm and sugarcane borer while negative control (#5) andHerculex®RW without Bt1 were susceptible to these two lepidopteras(Table 8). All events were consistent in tolerance to both fall armywormand sugarcane (Table 8). Transgenic event #20 was susceptible to bothlepidopteras possibly due to lack of Bt1 expression even with thepresence of Bt1 gene.

TABLE 8 In vitro fall armyworm and sugarcane borer bioassay resultsusing T₀ PHR03 plantlets Condition of Construct Bt1 gene Insect Insectleaves (0% Insect event # presence # alive # dead to 100%) Condition ofinsects Fall 26500 (#5) − 14 1 70% eaten Survivors premolt to IIIarmyworm Bt1 (#8) + 0 15  2% eaten All dead neonates Bt1 (#51) + 0 15 4% eaten All dead neonates Bt1 (#12) + 4 11  8% eaten Survivors still1sts Bt1 (#20) + 11 4 65% eaten Survivors premolt to III Herculex ® RW −13 2 85% eaten Survivors premolt to III Construct Bt1 gene Stalk Insectevent # presence holes Condition of leaves (0% to 100%) Sugarcane 26650(#5) − 10 Plant dead, stalk collapsed borer Bt1 (#8) + 2 Plant healthyBt1 (#51) + 6 Plant alive Bt1 (#12) + 6 Plant dying Bt1 (#20) + 8 Plantdying, stalk collapsed Herculex ® RW − ? Plant dead, stalk completelycollapsed unable to count *Some feeding on leaves, but neonates ofsugarcane borer borrowed into stalks within an hour and survivors couldnot be scored live/dead/.

Example 8 In Vitro Bioassay Using Multiple Insects

This innovative in vitro bioassay using plantlets provides a novelmethod to analyze effectiveness of gene constructs using both damages tothe plant as well as development of insect. Until recently, we have onlyperformed this bioassay using one insect at a time; however, a multipleinsect test has been successfully implemented in this example. Toprepare plantlets for this system, the whole plantlet was carefullyextracted from the MSB rooting medium with roots intact and with as muchagar off roots as possible and soaked for 5 minutes in a MSA+1.5% PPMsolution. A wire grid might be used to weigh down the plantlet in thesolution. After 5 min, the plantlet was carefully taken out and placedin a phytatray container containing 3 pieces of filter paper with 3 mLof MSA+1.5% PPM solution. The roots were spread out and the root/leafwas cut down if necessary so that all plantlets were equivalent. If onlyusing one type of insect for infestation, the plantlets were infestedand scored according to protocol about one week later. Multiple insectscould also be used. If the feeding source of the insects was different,i.e. one type fed on roots whereas the other fed on leaves, they couldbe infested together on the same day. When using multiple insects, theycould also be infested separately depending on how soon results showed,i.e. leaf-eating insect (lepidoptera) on one day and root-eating insectthree days later.

Table 9 demonstrates in vitro bioassay results using multiple insects inT_(o) quality event corn plantlets; all quality events contain Bt1 genefor lepidopteran resistance and Bt2 and Bt3 genes for rootwormresistance. All transgenic events showed resistance to both FAW and WCRWwhile negative control plantlets were susceptible to both insects (Table9). Infestation of both FAW and WCRW together was very efficient inscreening of transgenic events resistant to both insects compared withinfestation of each insect separately. The surviving plantlets weretransplanted to soil for additional insect bioassay, further molecularassay and grown to maturity to harvest seed. This bioassay schemeprovides an efficient and time-saving pre-screening system fortransgenic events (FIG. 1).

TABLE 9 In vitro bioassay using multiple insects in T₀ PHR03 plantletsConstruct Insect treatment (event #) Condition of leaves Condition ofinsects WCRW + FAW* - control plant dead; entire stalk and roots 5 FAWalive (III instars), (#623) destroyed (unclear if FAW ate roots as noWCRW found well) QE #8 no stalk damage - healthy plant; tiny no liveFAW, no growth pinhole damage (very slight), no root of WCRW damage QE#76 no stalk damage - healthy plant; 5% 2 FAW alive (II instars), damageto leaves by FAW, small WCRW alive but stunted damage to roots FAW−>WCRW** - control plant dying; stalk damage, 50% leaf 8 FAW alive (IIIinstars), (#623) eaten, no roots eaten by FAW one rootworm visible QE #8healthy plant; no leaf damage no live FAW, WCRW not on roots(repellency?) QE #76 healthy plant; no leaf damage no live FAW, WCRW onroots but stunted FAW−> WCRW* - control plant dead; nothing left 4 FAWalive (III instars) (#623) QE #76 healthy plant; 5% leaf damage from 1FAW (I), little WCRW FAW, no root damage yet mortality QE #105 plantcollapsing, browning; 10% leaf 3 FAW (II), some WCRW damage, minor rootdamage mortality QE #119 plant alive, green; 15% leaf damage, no 3 FAW(II), no WCRW root damage mortality FAW + WCRW** - control plant dead;stalk left, no roots, 70% leaf 3 FAW (III) +′ 1 eaten, 1 (#623) damageWCRW QE #76 healthy plant; no leaf damage, some 0 FAW, some WCRW rootdamage mortality QE #105 alive but collapsed; 20% leaf damage, 4 FAW(II), some WCRW no root damage mortality QE #119 plant alive; 2 FAW(II), ~50% 30% leaf damage, no root damage WCRW mortality WCRW−>FAW*** -control plant dead; stalk only left, major root 6 FAW (3 III, 3 II), 3(#623) damage, 70% leaf damage WCRW (II) QE #76 Alive; 20% leaf damage,minor root 3 FAW (I), 0 WCRW feeding QE #105 alive but collapsed; 5%leaf damage, no 6 FAW (I), 0 WCRW root damage QE #119 plant alive;pinhole leaf damage, no 2 FAW (I), 70% WCRW root damage mortality *Both25 neonates of western corn rootworm (WCRW) and 10 neonates of fallarmyworm (FAW) were infested together at the same time. **10 neonates ofFAW were infested first and 4 days later 25 neonates of WCRW wereinfested. ***25 neonates of WCRW were infested first and 6 days later 10neonates of FAW were infested.

Example 9 In Vitro Insect Bioassay for Promoter Testing

This example illustrates the application of the in vitro lepidopteraninsect bioassay system to test different promoters do drive theexpression of the Bt gene. Two constructs were used for corntransformation: one containing Bt1 driven by the maize ubiquitinpromoter (Ubi1-Bt1) and another driven by the banana streak viruspromoter (BSV TR-Bt1). Plantlets were regenerated from transgenic eventsand leaf punches were harvested for copy # assay by qPCR. Single copyevents were used for in vitro insect bioassay.

Under a laminar flow hood, 10 one-day-old larvae of fall armyworm (FAW)were transferred to the Phytatray containing the transgenic plantletusing a sterile disposable inoculating loop. Prior to infestation, aminimal amount of plant nutrients and 1.5% PPM was applied to the 3sterile filter papers on which the plantlet was placed. Metal meshscreen was placed on the roots of the plantlet to insure good contactbetween the roots and the plant nutrients on the filter paper ifnecessary. The Phytatrays were then placed in an incubator at 26° C. ina 16-hr light phase for 3-7 days prior to scoring the plants and larvaefor plant damage and larval development. Plants were observed todetermine those expressing Bt to kill or stunt FAW compared to thecontrol which were not expressing toxin.

Table 10 demonstrates the effect of Bt 1 expression driven by 2different promoters in corn plantlets on resistance to FAW. Alltransgenic events showed good resistance to fall armyworm, but thedegree of insect resistance was higher with the maize ubiquitin promoterthan banana streak virus promoter (Table 10). Bt1 expression of eachevent will be measured by ELISA or western blot analysis.

TABLE 10 Promoter test in T₀ PHR03 plantlets using in vitro insectbioassay Condition of leaves Promoter Construct event # (0% to 100%)Condition of insects -Control 26500 (#5) 70-100% eaten Survivors premoltto III Maize UbiI-Bt1 (#8) 0% eaten All dead neonates ubiquitin UbiI-Bt1(#12) 5% pinhole damage All dead neonates UbiI-Bt1 (#18) 2% eaten Alldead neonates UbiI-Bt1 (#47) 0% eaten All dead neonates UbiI-Bt1 (#51)4% eaten All dead neonates Banana streak BSV TR-Bt1 (#12) 8% eatenSurvivors still 1sts virus BSV TR-Bt1 (#76) 5% eaten Survivors still1sts BSV TR-Bt1 (#82) 0% eaten, stalk All dead neonates damage BSVTR-Bt1 (#105) 10% eaten 3 survivors to II BSV TR-Bt1 (#116) Pinholedamage All dead neonates BSV TR-Bt1 (#119) 15% eaten 3 survivors to II

Example 10 In Vitro Insect Bioassay Using Transgenic Green RegenerativeTissue Events

Our green tissue bioassay system also can provide a method forpre-screening of genes/promoters. The process is similar to the in vitrobioassay system that was described in Example 5, but instead of usingmaize plantlets, green regenerative tissues were used. Using all sterilematerials, two pieces of filter paper were placed into a Petri dish andabout 1.5 mL of MSA+1.5% PPM solution were pipetted onto the filterpaper. If there was excess solution, or not enough solution, solutionwas removed/added until the filter papers were evenly soaked but notdripping. Five good pieces of green tissue were selected and placed ontothe filter paper; good callus tissue is defined as a piece about 5 mm,and regenerable, compact, and green. Also, for all events beingscreened, there should be the same amount of green tissues and theyshould all be equivalent in quality and size. The Petri dish wasinfested with the insect which the gene that was being tested wasresistant against and the plate was sealed with parafilm to preventcontamination. Two days after infestation, additional solution was addedto keep the tissue healthy. After 6 to 12 days, tissue damage and insectgrowth/stunting/death were scored per scoring protocol.

Transgenic maize green tissue events transformed with 4 differentshuffled Bt variants were tested for WCRW resistance. As expected,negative control events were susceptible to WCRW and tissues becamebrown and some neonates grew to the healthy 2^(nd) instars (Table 11).Event #s 48 and 50 transformed with the shuffled Bt variant 14 showedgood efficacy to rootworms while event #95 transformed with the shuffledBt variant 1, event #36 transformed with the shuffled Bt variant 2 andevent #10 with the shuffled Bt variant 3 showed slightly to moderatelyresistant to rootworms. Clearer results could be obtained when thetissues were maintained for longer than 2 weeks. Thus, this green tissuebioassay system can be used for early screening of transgenic events.

TABLE 11 WCRW bioassay using T₀ PHR03 green regenerative tissuesConstruct Event # Tissue Damage Insect growth/healthiness 24600 (-control) 6 +++, brown healthy 26650 (- control) 1 +++, brown healthyShuffled Bt variant 1 34 ++(+), brown healthy 59 +++, brown healthy 95+, most of them green several dead/stunted Shuffled Bt variant 2 36+(+), most of them green several stunted Shuffled Bt variant 3 9 ++(+),brown healthy 10 +(+), some green some stunting Shuffled Bt variant 14 4++(+), brown healthy 48 0, green mortality and stunting 50 (+), greenmortality but some healthy Tissue damage and insectgrowth/stunting/death were scored 6 days after WCRW infestation.

Example 11 In Vitro Bioassay Using Different Explants for InsectInfestation

Different types of tissue were used for in vitro insect bioassayfollowing the similar sterilization and culture protocol used for the invitro insect bioassay protocol described above. Roots, leaves, callus-and green tissue-derived plantlets, and mature seed- and immatureembryo-derived seedlings were used as target explants for insectinfestation. Plantlets and germinating seedlings were, in general, bestfor in vitro insect bioassay using both rootworms and lepidopteras(Table 12).

TABLE 12 In vitro insect bioassay efficiency using different invitro-derived tissue types Germinating Germinating Green seedling fromseedling from Tissue type regenerative Regenerated mature seed w/immature seed Insect Root Leaf tissue Callus plantlet w/ root root w/root Rootworm +++ + ++(+) n.t. +++ +++ +++ Lepidoptera ++ +++ +++ n.t.+++ +++ +++ *n.t.: not tested

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A method for efficiently testing a polynucleotide of interest for aphenotype in roots comprising: a) regenerating green tissue that istransgenic for at least one polynucleotide of interest into one or moretransgenic plantlets, wherein the one or more transgenic plantletscomprise at least one transgenic root; and b) determining at least onephenotype of the transgenic root.
 2. The method of claim 1, furthercomprising subjecting the transgenic root to a pest or pathogen.
 3. Themethod of claim 1, wherein the phenotype is increased root size,increased overall root mass, altered root architecture, increasedexpression level of mRNA or protein, increased biochemical content,increased tolerance to stress, increased resistance to a pest, increasedresistance to an insecticide, increased yield, or increased nitrogen useefficiency as compared to the corresponding phenotype of a control,wherein the polynucleotide of interest has not been introduced into thecontrol.
 4. The method of claim 1, wherein the at least one transgenicroot is isolated from the plantlet prior to determining the at least onephenotype.
 5. The method of claim 1, further comprising producing atransgenic plant from the green tissue.
 6. The method of claim 1,further comprising growing the transgenic plantlet into a transgenicplant.
 7. The method of claim 1, further comprising rooting the plantleton medium.
 8. The method of claim 7, wherein the medium is medium gelledwith agar or an agar substitute.
 9. The method of claim 1, wherein theplantlet is a monocot plantlet.
 10. The method of claim 1, wherein thegreen tissue is obtained by transforming an explant from a monocot andsubjecting the explant to green tissue initiation medium for a time andunder conditions sufficient to initiate growth from the explant, therebyproducing green tissue.
 11. The method of claim 10, wherein the explantcomprises an embryo, green tissue, callus, leaf, meristem, seedling,seed, stem, shoot, node, leaf base, or root.
 12. The method of claim 1,further comprising regenerating transgenic green tissue into one or moretransgenic plantlets by contacting the green tissue with a rootingmedium that induces root formation for a time and under conditionssufficient to initiate root growth from the green tissue, therebyproducing a plantlet.
 13. The method of claim 1, wherein determining atleast one phenotype of the transgenic root occurs under sterileconditions.
 14. A method of making a root assay comprising: a)contacting green tissue with a first rooting medium gelled with agar oran agar substitute to produce a plantlet having at least one transgenicroot; b) removing the root from the medium; and c) contacting the rootwith a second rooting medium in the absence of agar or an agarsubstitute to produce a plurality of roots.
 15. The method of claim 14,further comprising regenerating a plurality of transgenic plantlets fromthe green tissue.
 16. The method of claim 14, placing the at least onetransgenic root of the plantlet in a culture dish.
 17. The method ofclaim 14, contacting the roots with the second rooting medium, whereinthe second rooting medium comprises a biocide.
 18. The method of claim14, contacting the roots with the second rooting medium using anabsorbent material.
 19. The method of claim 14, wherein the green tissueis obtained by transforming an explant from a monocot and subjecting theexplant to green tissue initiation medium for a time and underconditions sufficient to initiate growth from the explant, therebyproducing green tissue.
 20. The method of claim 19, wherein the explantcomprises an embryo, green tissue, callus, leaf, meristem, seedling,seed, stem, shoot, node, leaf base, or root.
 21. The method of claim 14,further comprising regenerating transgenic green tissue into one or moretransgenic plantlets by contacting the green tissue with a first rootingmedium that induces root formation for a time and under conditionssufficient to initiate root growth from the green tissue, therebyproducing a plantlet.
 22. The method of claim 14, wherein the roots areprepared under substantially sterile conditions.
 23. The method of claim14, further comprising subjecting the root to a chemical, pest, orpathogen.
 24. The method of claim 23, further comprising sterilizing thepest prior to contacting the root.
 25. The method of claim 23, furthercomprising feeding the pest prior to infesting the pest on thetransgenic roots.
 26. The method of claim 14, wherein the assay isproduced within about 4 to 14 days.
 27. The method of claim 14, furthercomprising growing the transgenic plantlets into plants.
 28. The methodof claim 27, further comprising obtaining transgenic seeds from thetransgenic plants.
 29. A method of assaying for insecticidal activity ona live root comprising: a) regenerating green tissue into one or moreplantlets comprising at least one live root; b) contacting the at leastone root of the plantlet with a rooting medium; c) exposing the root toone or more pests to infest the medium for infestation; wherein themedium and pest are substantially contamination-free; and d) determininga phenotype of the root.
 30. The method of claim 29, further comprisingregenerating green tissue transgenic for a polynucleotide of interestinto one or more transgenic plantlets comprising at least one livetransgenic root.
 31. The method of claim 29, wherein the live root isassayed for endogenous resistance to root damage by the pest.
 32. Themethod of claim 29, further comprising contacting the green tissue withrooting medium to induce root formation.
 33. The method of claim 29,wherein the rooting medium comprises a biocide.
 34. The method of claim29, contacting the root with the rooting medium using an absorbentmaterial.
 35. The method of claim 29, determining the phenotype of theroot by scoring the root for damage as compared to the phenotype of thecontrol.
 36. The method of claim 29, further comprising sterilizing thepest prior to contacting the root.
 37. The method of claim 29, furthercomprising feeding the pest prior to infesting the pests on thetransgenic roots.
 38. The method of claim 29, wherein the pest is fromthe order of lepidoptera, homoptera, heteroptera, or coleoptera.
 39. Themethod of claim 29, wherein the pest is of a developmental stagecomprising an egg, larva, instar, or adult.
 40. The method of claim 29,further comprising determining a phenotype of the pest.
 41. The methodof claim 40, wherein the phenotype is growth or mortality of the pest.42. The method of claim 41, further comprising determining the phenotypeby scoring the pest for mortality or stunted growth.
 43. The method ofclaim 42, wherein more than half of the pests are dead or have stuntedgrowth indicates the polynucleotide of interest is effective forcontrolling pest infestation or damage to the root or combinationsthereof.
 44. The method of claim 29, comprising observing the stem areaabove a crown, wherein the stem area darkens from pest damage ascompared to a control indicates that the root is not effective incontrolling pest infestation.
 45. The method of claim 29, wherein thegreen tissue is obtained by transforming an explant from a monocot andsubjecting the explant to green tissue initiation medium for a time andunder conditions sufficient to initiate growth from the explant, therebyproducing green tissue.
 46. The method of claim 45, wherein the explantcomprises an embryo, green tissue, callus, leaf, meristem, seedling,seed, stem, shoot, node, leaf base, or root.
 47. The method of claim 29,further comprising regenerating transgenic green tissue into one or moretransgenic plantlets by contacting the green tissue with a first rootingmedium that induces root formation for a time and under conditionssufficient to initiate root growth from the green tissue, therebyproducing a plantlet.
 48. The method of claim 29, further comprisingproducing a plurality of transgenic plantlets from the green tissue. 49.The method of claim 29, further comprising producing a plurality ofroots from the green tissue.
 50. The method of claim 29, furthercomprising placing the at least one root of the plantlet in a culturedish.
 51. The method of claim 29, wherein the plantlet has had itsleaves removed.
 52. The method of claim 29, wherein the root is intact.53. The method of claim 29, wherein the assay is completed within about4 to 14 days.
 54. The method of claim 29, further comprisingregenerating transgenic plantlets from the green tissue from which aroot has been assayed for its phenotype.
 55. A substantiallycontamination-free, root bioassay comprising: a monocot plantlet,wherein the plantlet comprises at least one live root in a culture dish,and wherein the dish comprises a rooting medium in contact with theroot.
 56. The assay of claim 55, wherein the plantlet and root aretransgenic for a polynucleotide of interest.
 57. The assay of claim 55,further comprising a plantlet having a plurality of roots.
 58. The assayof claim 55, further comprising a plurality of plantlets obtained fromthe green tissue.
 59. The assay of claim 55, wherein the rooting mediumcomprises a biocide.
 60. The assay of claim 55, wherein the assaycomprises a means for bringing the root into contact with the rootingmedium.
 61. The assay of claim 55, wherein the culture dish comprises asubstantially sterile absorbent material that contacts the rootingmedium and the roots.
 62. The assay of claim 55, wherein the assaycomprises a sterilized pest, pathogen, or chemical.
 63. The assay ofclaim 55, wherein the pest is from the order of lepidoptera, homoptera,heteroptera, or coleoptera.
 64. The assay of claim 55, wherein the pestis of a developmental stage comprising an egg, larva, instar, or adult.65. The assay of claim 62, wherein the chemical is a pesticide,insecticide, fungicide, or bactericide.
 66. The assay of claim 62,wherein the root is transgenic for a Bt gene and wherein the pest iswestern corn root worm (WCRW).
 67. A method of identifying a promoterhaving activity in the root comprising: a) regenerating green tissuetransgenic for a promoter of interest operably linked to apolynucleotide into one or more stably transformed transgenic plantlets,wherein the plantlets comprise at least one transgenic root; and b)determining whether the polynucleotide is expressed in root cells of theplantlet.
 68. The method of claim 67, determining whether thepolynucleotide is expressed preferentially in root cells of theplantlet.
 69. The method of claim 67, comprising determining whether thepolynucleotide is expressed preferentially in root cells of the plantletas compared to expression in cells of non-root tissues of the plantlet;wherein increased expression of the polynucleotide in root cells incomparison to expression of the polynucleotide in non-root cellsindicates that the promoter is preferentially expressed in root cells.70. The method of claim 67, wherein the at least one transgenic root isisolated from the plantlet prior to determining the expression of thepolynucleotide.
 71. The method of claim 67, further comprising producinga transgenic plant from the green tissue.
 72. The method of claim 67,further comprising growing the transgenic plantlet into a transgenicplant
 73. The method of claim 67, further comprising rooting theplantlet on medium.
 74. The method of claim 73, further comprisingrooting the plantlet on medium gelled with agar or an agar substitute.75. The method of claim 67, wherein the plantlet is a monocot plantlet.76. The method of claim 67, wherein the green tissue is obtained bytransforming an explant from a monocot and subjecting the explant togreen tissue initiation medium for a time and under conditionssufficient to initiate growth from the explant, thereby producing greentissue.
 77. The method of claim 76, wherein the explant comprises anembryo, green tissue, callus, leaf, meristem, seedling, seed, stem,shoot, node, leaf base, or root.
 78. The method of claim 67,regenerating transgenic green tissue into one or more transgenicplantlets by contacting the green tissue with a rooting medium thatinduces root formation for a time and under conditions sufficient toinitiate root growth from the green tissue, thereby producing aplantlet.
 79. The method of claim 67, wherein the polynucleotide encodesa marker polypeptide.